SUMMARY/ABSTRACT During development, growth cones (GCs) of diverse cortical or other projection neuron (PN) subtypes navigate complex extracellular environments to reach distant, subtype-specific targets. These axon-terminal structures must respond to substrate-bound and diffusible signals in a subtype- and stage/context-specific fashion to construct specific functional circuitry. The proposed work aims to deeply investigate the protein and RNA composition and function of growth cones in a subtype- and stage/context-specific fashion to address longstanding questions in the field of cortical circuit and other nervous system development. Recent studies strongly indicate that subcellular localization of specific molecular machinery to GCs might underlie the precise behaviors of these structures during circuit ?wiring.? While great progress has been made toward identifying diffusible and substrate-bound signals that guide axon growth, it is becoming increasingly clear that intracellular local growth cone biology underlies the distinct responses of specific neuronal subtypes at specific stages in specific contexts. Molecular determinants of these critical processes remain largely unstudied with respect to distinct neuronal subtypes under physiological conditions. Because most current knowledge of growth cone biology was identified in vitro, often with heterogeneous populations, access to subtype-specific growth cones in their native environment during normal development will substantially elucidate molecular bases of cortical circuit formation. Our laboratory has recently developed an innovative approach that enables high-throughput proteomic and transcriptomic investigation of GCs from fluorescently labeled subtype-specific cortical projection neurons. This approach has already yielded unanticipated and exciting new biological insight, which is now submitted for publication and under review. Building on the foundational work of our laboratory, I have independently reproduced all the experiments, demonstrating the feasibility of the proposed research in my own hands. GCs will be isolated from readily accessible, closely-related PN subtypes with distinct axonal trajectories at critical developmental stages to investigate whether and how subtype-specific GC molecular machinery might functionally enable specific subtypes to respond to extracellular cues in precise, characteristic fashions. In particular, I will isolate GCs from corticothalamic PN that implement an intrahemispheric corticofugal trajectory as well as from callosal PN at critical stages (pre- and post-midline crossing) during the development of interhemispheric corticocortical projections. The proposed experiments will generate unique knowledge about the molecular development of subtype-specific cortical neuron connectivity. These rigorous and unbiased data will elucidate subtype- (Aim 1) and stage-specific (Aim 2) growth cone biology in vivo; identify novel regulators of cortical circuit development, maintenance, and function (Aim 3); identify molecular mechanisms that might underlie development of neurodegenerative or neuropsychiatric disease; and potentially develop new experimental strategies to address fundamental questions of cortical circuit development (Aim 3).